The activation of proteins by post-translational modification represents an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. For example, protein phosphorylation plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes, as well as in proper immune function. In spite of the importance of protein modification, it is not yet well understood at the molecular level. The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, and second, that the technology necessary to unravel its complexity has not yet been fully developed.
The complexity of protein modification, including phosphorylation, on a proteome-wide scale derives from three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome encodes, for example, over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Each of these kinases phosphorylates specific serine, threonine, or tyrosine residues located within distinct amino acid sequences, or motifs, contained within different protein substrates. Most kinases phosphorylate many different proteins: it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases. See Graves et al., Pharmacol. Ther. 82: 111-21 (1999).
Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra. Oncogenic kinases such as ErbB2 and Jak3, widely expressed in breast tumors and various leukemias, respectively, transform cells to the oncogenic phenotype at least in part because of their ability to phosphorylate cellular proteins. Understanding which proteins are modified by these kinases will greatly expand our understanding of the molecular mechanisms underlying oncogenic transformation. Thus, the ability to identify modification sites, e.g. phosphorylation sites, on a wide variety of cellular proteins is crucially important to understanding the key signaling proteins and pathways implicated in disease progression, as well as critical biological processes such as the immune response.
The efficient identification of protein phosphorylation sites relevant to signal transduction has been aided by the recent development of a powerful new class of antibodies, called motif-specific, context-independent antibodies, which are capable of specifically binding short, recurring signaling motifs comprising one or more modified (e.g. phosphorylated) amino acids in many different proteins in which the motif recurs. See U.S. Pat. No. 6,441,140, Comb et al. Many of these powerful new antibodies are now available commercially. See Cell Signaling Technology, Inc. 2003-04 Catalogue. More recently, a powerful new method for employing such motif-specific antibodies in immunoaffinity techniques coupled with mass spectrometric analysis to rapidly identify modified peptides from complex biological mixtures has been described. See U.S. Patent Publication No. 20030044848, Rush et al.). Such techniques will enable the rapid elucidation of protein activation and phosphorylation events underlying diseases, like cancer, that are driven by disruptions in signal transduction, as well as those underlying critical biological processes such as the immune response.
The transmission of intracellular signaling resulting from binding of the T-lymphocyte receptor (T-cell receptor) to foreign antigen presented with the major histocompatability complex (MHC) on antigen presenting cells (APCs) is a process critical to the generation of a proper immune response in mammals. Antigen-specific T-cell binding via the T-cell receptor results in a kinase-mediated signaling cascade leading to cell-specific proliferation of the activated T-cells, and their participation in the immune response against foreign antigens and cells. Defects in T-cell signaling have been associated with T-cell acute lymphocytic leukemias. See Blume-Jensen et al., Nature 411: 355-365 (2001) (describing T cell receptor beta gene translocation next to the gene encoding the Lck tyrosine kinase gene, resulting in presumably constitutive activation of Lck).
T-cell receptor-induced signaling is mediated through a variety of second messengers, protein kinases and phosphatases, and other enzymes and intermediates. It is now known that binding of the human T-cell receptor to specific antigen-MHC complex results in the activation and/or recruitment of the Src-family kinases, Lck and Fyn, which in turn phosphorylate two critical tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR-ζ invariant chain of the TCR complex. See, e.g. Mustelin et al., Biochem J. 371: 15-27 (2003); Pitcher et al., Trends in Immunol., 24: 554-560 (2003). This process may also involve the exclusion of protein tyrosine phosphatases that would down-regulate Lck and Fyn, as well as the exclusion of Csk kinase, which negatively regulates Lck and Fyn by phosphorylation at a conserved C-terminal tyrosine (Tyr505 in Lck and Try528 in Fyn). See Mustelin et al., supra.
Phosphorylation of the ITAMs renders them high-affinity ligands for the ZAP-70 kinase, which is selectively recruited to the activated receptor complex, and (along with the kinase Syk) is subsequently activated by phosphorylation at tyrosine 493 (Tyr493) by Lck kinase. See Mustelin et al., Pitcher et al., supra. Following its activation, ZAP-70, along with Syk, in turn phosphorylates other key downstream adaptor proteins (such as LAT) and effector proteins (such as SLP-76). Further, certain phosphorylated tyrosine sites in activated ZAP-70 provide key docking sites for SH-2 domain-containing effector proteins like Lck and Cbl, which participate in a complex cascade—involving Ca2+/InsP3, Ras/Raf/ERK and RhoA pathways, ultimately leading to gene regulation and cell proliferation. See Mustelin et al., Pitcher et al., supra.
Although some of the signaling proteins and phosphorylation sites involved in proper T-cell receptor signaling have been identified, a clear picture of the precise proteins and phosphorylation sites involved in propagating this essential biological signal remains to be developed. For example, SHP1 phosphatase and Fyn kinase may be involved in the signaling cascade, but their precise role and substrates are unknown. See Mustelin et al., supra. Other Src-family protein tyrosine kinases, including the Tec-related kinases, Itk/Emt and Txk/Rlk, appear to be involved as well, but their precise role and substrates remains to be determined. Accordingly, the small number of T-cell receptor signaling pathway-related phosphorylation sites that have been identified to date do not facilitate a complete and accurate understanding of how this important biological signal is propagated. Indeed, it has recently been concluded that a major remaining challenge in T-cell biology is more precisely define the contribution of particular signaling molecules involved in the T-cell signaling, and to better understand the interplay between signaling molecules and pathways involved. See Mustelin et al., supra.
Accordingly, there is a continuing need to unravel the molecular mechanisms of T-cell receptor signaling by identifying the downstream signaling proteins mediating the cascade leading to proliferation of activated T-cells and their participation in the immune response. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho-specific antibodies and AQUA peptides, to detect and quantify them remains particularly important to advancing our understanding of the biology of the critical T-cell signaling process. In turn, such advances would lead to a better understanding of diseases, such as T-cell acute lymphocytic leukemias, involving aberrant T cell signaling. See Blume-Jensen et al., supra.